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目的:建立专属、灵敏的液相色谱-串联质谱法(LC-MS/MS)法测定人血浆中的雷洛昔芬,并将方法应用于雷洛昔芬两种制剂的人体生物等效性研究。方法:血浆样品中加入200μLβ-葡萄糖苷酸酶于37℃水浴孵化10 h后采用液-液萃取法预处理。Agilent Zorbax SB-C18色谱柱(150 mm×4.6 mm,5μm)进行分离,流动相为甲醇-醋酸铵(5 mmol.L-1)-甲酸(65∶35∶0.1),流速为0.6 mL.min-1。大气压化学电离源,多反应监测方式(MRM)进行正离子检测,定量分析离子对为m/z 474→m/z 112(雷洛昔芬)和m/z 478→m/z116(内标d4-雷洛昔芬)。临床试验采用随机双交叉设计,24例健康男性受试者空腹单次口服60 mg雷洛昔芬受试制剂或参比制剂,LC-MS/MS法测定血浆雷洛昔芬浓度,计算有关药代动力学参数并进行生物等效性评价。结果:雷洛昔芬定量方法线性范围为0.20～250 ng.mL-1,定量下限为0.20 ng.mL-1,日内、日间精密度(RSD)均小于11.2%,准确度(RE)在-4.0%～1.3%之间。两种制剂的AUC0～120无显著性差异,Cmax和Tmax有显著性差异。结论:本方法专属、灵敏,适用于雷洛昔芬两种制剂的人体生物等效性评价。两种雷洛昔芬制剂吸收程度相似,但受试制剂Cmax显著降低,Tmax延长,未体现出其分散片的特点。
OBJECTIVE: To establish a specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for the determination of raloxifene in human plasma and to apply the method to the bioequivalence of raloxifene the study. Methods: 200μL β-glucuronidase was added to the plasma samples and incubated in a water bath at 37 ℃ for 10 h before liquid-liquid extraction. Agilent Zorbax SB-C18 column (150 mm × 4.6 mm, 5 μm) with a mobile phase of methanol-ammonium acetate (5 mmol·L -1) -carboxylic acid (65:35:0.1) at a flow rate of 0.6 mL · min -1. Atmospheric pressure chemical ionization source, multiple reaction monitoring (MRM) for positive ion detection, quantitative analysis of ion pairs m / z 474 → m / z 112 (raloxifene) and m / z 478 → m / z116 - raloxifene). A randomized crossover design was used in clinical trials. Twenty-four healthy male subjects were dosed orally with either 60 mg raloxifene or reference preparations on a fasting basis, and the plasma raloxifene concentration was determined by LC-MS / MS, Substitution kinetic parameters and bioequivalence assessment. Results: The linear range of raloxifene was 0.20-250 ng.mL-1 with a lower limit of quantitation of 0.20 ng.mL-1. The intra-and inter-day RSD was less than 11.2%, and the accuracy (RE) -4.0% ~ 1.3%. There was no significant difference in AUC0 ~ 120 between the two preparations, but there was a significant difference between Cmax and Tmax. Conclusion: This method is specific and sensitive and is suitable for the bioequivalence assessment of raloxifene in two kinds of human body. The two raloxifene preparations absorbed similar levels, but the test preparation Cmax significantly reduced, Tmax extended, did not reflect the characteristics of its dispersible tablets.